This proposal examines the role of proteolipids in biologic mineral formation and the mechanism of their action in situ. Matrix vesicle proteolipid (MVP) produced by rat chondrocytes in culture is used as a model system to test the hypothesis that MVP is a site of initial hydroxyapatite formation and that changes in the membrane environment, such as those produced by the action of vitamin D, will alter its functional properties. A model membrane system will be used to mimic the membrane environment in vitro. MVP will be purified by differential extraction in organic solvents of varying polarities, reverse phase and chromatofocusing HPLC. Heterogeneity will be assessed by ISF and SDS-PAGE. Polyclonal antibodies generated to MVP will be affinity purified and then used to affinity purify specific subsets of MVP, to characterize multimeric aggregates, to purify mRNA for individual proteolipids, and to chart MVP synthesis and incorporation into matrix vesicles. MVP will be incorporated into planar bilayers of known composition and ion transport compared to that of the intact matrix vesicle membrane. The ability of specific functional units to support hydroxyapatite formation will be tested in vitro. To examine the effect of regulation of the membrane microenvironment on MVP structure and function in culture and in vitro, reserve zone (RC) or growth region (GC) chondrocytes will be incubated with 1,25(OH)2D3 or 24R,25(OH)2D3. Changes in matrix vesicle and plasma membrane fluidity will be assessed using flourescence spectroscopy; membrane structure and function monitored by enzymatic activity (alkaline phosphatase, 5' nucleotidase, Na+/K+ ATPase) and phospholipid composition. Incorporation of vitamin D into the membrane will be measured using 3H-labeled metabolites. Phospholipid turnover will also be assessed as acylation and hydrolysis of radiolabeled fatty acids. Synthesis of MVP and its incorporation into matrix vesicles will be charted using specific antibodies. Membrane and MVP function will be measured using planar bilayer membranes or by in vitro hydroxyapatite formation.